Broad, long-term Objectives. To elucidate, at a molecular level: l) the mechanism of catalysis, 2) specificity determinants for substrates and coenzyme, and 3) why phosphorylation down regulates activity of the 4- electron oxidoreductase HMG-CoA reductase. Specific Aims. 1) Identify active site residues which function in catalysis, substrate recognition, or maintaining conformation. 2) Provide physical evidence in support of the P.I. 's proposed novel mechanism for regulation of activity that accompanies phosphorylation of a unique serine, namely, charge-charge interaction with the catalytic histidine. 3) Generate mutant proteins to facilitate visualization of the three- dimensional structures of the "third domain" of the P. mevalonii enzyme. 4. Overproduce homogeneous hamster HMG-CoA reductase to determine conditions for crystallization and ultimate structure determination. 5) Identify regions of bacterial and hamster HMG-CoA reductase which participate in substrate recognition and binding. 6) Characterize an archaebacterial HMG-CoA reductase. Health Relatedness. The rate-limiting enzyme of human cholesterogenesis, HMG-CoA reductase represents an established target for chemotherapy of hypercholesterolemias. Benefits from the proposed research include improved understanding of the mechanisms of catalysis and of phosphorylation-mediated control of HMG-CoA reductase activity. Practical benefits include enhanced capability for rational design of drugs which inhibit HMG-CoA reductase activity. The compounds in wide use to lower cholesterol levels in hypercholesterolemic individuals are widely held to be active site-directed inhibitors. However, recent work from the P.I. 's laboratory has located the regulatory serine at the active site, and has shown that phosphorylation blocks the function of the catalytic histidine. Existing and future drugs thus might act by interfering with dephosphorylation of the regulatory serine. Research Design and Methodologies. The research will employ HMG-CoA reductases from all three phylogenetic kingdoms: eukaryotes, eubacteria, and the archae. Inter-kingdom chimeric enzymes will also be generated and characterized. Selection of amino acids to be mutated will guided by the now available 3 A resolution crystal structure of P. mevalonii HMG-CoA reductase. Mutant enzymes will be overexpressed, purified, and their enzymic and biophysical properties characterized. Methods to be employed encompass molecular biological, biochemical, and biophysical techniques: site-directed mutagenesis and PCR; overexpression; enzyme purification; enzymic and physical characterization of mutant enzymes; reconstitution of active enzymes by co-expression of mutually complementary inactive mutant enzymes; construction of chimeric enzymes; crystallographic determination of three-dimensional structures.